Emerging mechanisms of telomerase reactivation in cancer
Authors: Shalu Sharma, Shantanu Chowdhury
Journal: Trends in cancer (2022)

Ninety percent of cancers hyperactivate telomerase to induce continuous cell proliferation. Two clinically significant noncoding point mutations within the human telomerase reverse transcriptase (hTERT) core promoter have been found causal for telomerase hyperactivation across >40% of all cancers.G>A transitions at position −124/−146 from the transcription start site on the mutant hTERT promoter were found to generate novel E26 transformation-specific (ETS) factor binding sites. Several studies have unraveled different ETS-associated mechanisms causing transcriptional hyperactivation of telomerase owing to the mutations.The role of chromatin looping in telomerase reactivation has also come to light in this context; we summarize all such known mechanisms and present potential connections between these.Recent findings demonstrating the role of telomere-binding proteins and G-quadruplexes in telomerase regulation unravel another mode of potentially telomere length-dependent telomerase reactivation across cancer cells. In this review, we present the crosstalk between all mechanisms known in hyperactivating telomerase in hTERT promoter mutant cancers. Mutations in the promoter of human telomerase reverse transcriptase (hTERT) result in hyperactivation of hTERT. Notably, all mutations are G>A transitions, frequently found in a wide range of cancer types, and causally associated with cancer progression. Initially, the mutations were understood to reactivate hTERT by generating novel E26 transformation-specific (ETS) binding sites. Recent work reveals the role of DNA secondary structure G-quadruplexes, telomere binding factor(s), and chromatin looping in hTERT regulation. Here, we discuss these emerging findings in relation to the clinically significant promoter mutations to provide a broader understanding of the context-dependent outcomes that result in hTERT activation in normal and pathogenic conditions. Mutations in the promoter of human telomerase reverse transcriptase (hTERT) result in hyperactivation of hTERT. Notably, all mutations are G>A transitions, frequently found in a wide range of cancer types, and causally associated with cancer progression. Initially, the mutations were understood to reactivate hTERT by generating novel E26 transformation-specific (ETS) binding sites. Recent work reveals the role of DNA secondary structure G-quadruplexes, telomere binding factor(s), and chromatin looping in hTERT regulation. Here, we discuss these emerging findings in relation to the clinically significant promoter mutations to provide a broader understanding of the context-dependent outcomes that result in hTERT activation in normal and pathogenic conditions. Healthy human cells determine cellular proliferation and lifespan in a lineage-dependent manner. This process is achieved through a delicately balanced and tightly coordinated regulation of cellular proliferation and aging. While premature aging can result in pathologies such as progeria (see Glossary), Alzheimer disease, and alopecia, uncontrolled cell proliferation can cause cancer [1.Lander A.D. et al.Cell lineages and the logic of proliferative control.PLoS Biol. 2009; 7e15Crossref Scopus (234) Google Scholar,2.Duronio R.J. Xiong Y. Signaling pathways that control cell proliferation.Cold Spring Harb. Perspect. Biol. 2013; 5a008904Crossref PubMed Scopus (217) Google Scholar]. Consequently, decades of scientific exploration continue to investigate the underlying mechanisms of proliferation. Among these mechanisms is the role of telomeres. Telomeres are G-rich repetitive sequences at the ends of chromosomes that are replicated by the specialized reverse transcriptase telomerase, which comprises the catalytically active reverse transcriptase TERT and its RNA template TERC. In human normal somatic cells, hTERT is maintained in a closed state such that cells lack telomerase and are unable to maintain telomeres following cell division. As a result, telomeres shorten gradually until critically short telomeres trigger replicative senescence or apoptosis. Most cancer cells, however, experience reactivation or hyperactivation of hTERT, which disturbs the normal events leading to telomere shortening, thereby inducing uncontrolled cell proliferation [3.Shay J.W. et al.Telomerase and cancer.Hum. Mol. Genet. 2001; 10: 677-685Crossref PubMed Scopus (355) Google Scholar, 4.Shay J.W. Role of telomeres and telomerase in aging and cancer.Cancer Discov. 2016; 6: 584-593Crossref PubMed Scopus (353) Google Scholar, 5.Shay J.W. Bacchetti S. A survey of telomerase activity in human cancer.Eur. J. Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2377) Google Scholar, 6.Akincilar S.C. et al.Reactivation of telomerase in cancer.Cell. Mol. Life Sci. 2016; 73: 1659-1670Crossref PubMed Scopus (115) Google Scholar, 7.Shay J.W. Wright W.E. Senescence and immortalization: role of telomeres and telomerase.Carcinogenesis. 2005; 26: 867-874Crossref PubMed Scopus (542) Google Scholar]. Regulation of hTERT activity was broadly understood to be governed by holoenzyme assembly, cellular localization, and transcriptional control. Point mutations within the hTERT promoter were found to be prevalent in a significant proportion of patients with different cancer types, including ~83% of glioblastoma cases, 70% of hepatocellular carcinoma cases, 85% of bladder cancer cases, up to 75% of melanoma and non-melanoma skin cancer cases, 80% of urinary tract cancer cases, and 50% of thyroid cancer cases (Figure 1). In all cases, these mutations were G>A transitions and were found to be causally associated with telomerase hyperactivation, poor patient survival, and disease recurrence [8.Liu H.-Y. et al.Conformation selective antibody enables genome profiling and leads to discovery of parallel G-quadruplex in human telomeres.Cell Chem. Biol. 2016; 23: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 9.Chiba K. et al.Cancer-associated TERT promoter mutations abrogate telomerase silencing.eLife. 2015; 4e07918Crossref Scopus (175) Google Scholar, 10.Liu R. Xing M. Diagnostic and prognostic TERT promoter mutations in thyroid fine-needle aspiration biopsy.Endocr. Relat. Cancer. 2014; 21: 825-830Crossref PubMed Scopus (59) Google Scholar, 11.Bullock M. et al.ETS factor ETV5 activates the mutant telomerase reverse transcriptase promoter in thyroid cancer.Thyroid. 2019; 29: 1623-1633Crossref PubMed Scopus (17) Google Scholar, 12.Myung J.K. et al.TERT promoter mutations and tumor persistence/recurrence in papillary thyroid cancer.Cancer Res. Treat. 2016; 48: 942-947Crossref PubMed Scopus (13) Google Scholar, 13.Crescenzi A. et al.Preoperative assessment of TERT promoter mutation on thyroid core needle biopsies supports diagnosis of malignancy and addresses surgical strategy.Horm. Metab. Res. 2016; 48: 157-162PubMed Google Scholar, 14.Sahm F. et al.TERT promoter mutations and risk of recurrence in meningioma.J. Natl. Cancer Inst. 2016; 108: djv377Crossref PubMed Scopus (220) Google Scholar, 15.Rachakonda P.S. et al.TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17426-17431Crossref PubMed Scopus (237) Google Scholar, 16.Landa I. et al.Frequent somatic TERT promoter mutations in thyroid cancer: higher prevalence in advanced forms of the disease.J. Clin. Endocrinol. Metab. 2013; 98: E1562-E1566Crossref PubMed Scopus (298) Google Scholar, 17.Melo M. et al.TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas.J. Clin. Endocrinol. Metab. 2014; 99: E754-E765Crossref PubMed Scopus (367) Google Scholar, 18.Razis E. et al.Is there an independent role of TERT and NF1 in high grade gliomas?.Transl. Oncol. 2020; 13: 346-354Crossref PubMed Scopus (7) Google Scholar]. Understanding how promoter mutations contribute to hTERT activation has mostly focused on the novel activator protein binding site(s) that is generated as a result of the mutations (reviewed in [19.Ramlee M.K. et al.Transcription regulation of the human telomerase reverse transcriptase (hTERT) gene.Genes (Basel). 2016; 7: 50Crossref PubMed Scopus (103) Google Scholar,20.Liu T. et al.Cancer-specific telomerase reverse transcriptase (TERT) promoter mutations: biological and clinical implications.Genes (Basel). 2016; 7: 38Crossref Scopus (88) Google Scholar]). Recent findings, however, add new dimensions, including the role of hTERT promoter mutations in mediating chromatin looping; interaction of telomeres with the hTERT loci; and the effect of non-duplex DNA secondary structures, G-quadruplexes, formed within the hTERT promoter. These emerging findings and the crosstalk among the apparently distinct mechanisms in possibly context-dependent ways might significantly contribute to our understanding of hTERT regulation in neoplastic transformation, in addition to pluripotency, aging, and T-cell activation. In this review, we detail the mechanisms of hTERT hyperactivation and highlight emerging possibilities. hTERT promoter mutations were discovered to occur at several positions, including −57 bp (1295161T>G), −124 bp (1295228 G>A), −146 bp (1295250G>A), and −245 bp (rs2853669 G/A) upstream of the translation start site [19.Ramlee M.K. et al.Transcription regulation of the human telomerase reverse transcriptase (hTERT) gene.Genes (Basel). 2016; 7: 50Crossref PubMed Scopus (103) Google Scholar,21.Horn S. et al.TERT promoter mutations in familial and sporadic melanoma.Science. 2013; 339: 959-961Crossref PubMed Scopus (1245) Google Scholar, 22.Huang F.W. et al.Highly recurrent TERT promoter mutations in human melanoma.Science. 2013; 339: 957-959Crossref PubMed Scopus (1291) Google Scholar, 23.Killela P.J. et al.TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 6021-6026Crossref PubMed Scopus (978) Google Scholar] (Figure 2). Among these, −124G>A and −146G>A are the most common and are mostly monoallelic [24.Stern J.L. et al.Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.Genes Dev. 2015; 29: 2219-2224Crossref PubMed Scopus (129) Google Scholar,25.Huang F.W. et al.TERT promoter mutations and monoallelic activation of TERT in cancer.Oncogenesis. 2015; 4e176Crossref Scopus (51) Google Scholar]. Both of these mutations lead to generation of a 11-bp sequence, CCCGGAAGGGG, that is similar to the ETS factor binding motif [15.Rachakonda P.S. et al.TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17426-17431Crossref PubMed Scopus (237) Google Scholar]. Of the 27 ETS binding factors known, GABP, ETS1, and ETV3 were screened as potential reactivators of hTERT expression. The effect of GABP on hTERT hyperactivation was most drastic in glioblastomas with the hTERT promoter mutation(s). In addition, GABP binding on the mutant hTERT promoter resulted in direct transcriptional activation of hTERT expression [26.Bell R.J.A. et al.The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer.Science. 2015; 348: 1036-1039Crossref PubMed Scopus (343) Google Scholar]. Similar effects of GABPalpha/beta (GABPA/B1)-mediated telomerase reactivation are also observed in melanoma, hepatocellular carcinoma, neuroblastoma, and bladder cancer [24.Stern J.L. et al.Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.Genes Dev. 2015; 29: 2219-2224Crossref PubMed Scopus (129) Google Scholar,27.Akıncılar S.C. et al.Long-range chromatin interactions drive mutant TERT promoter activation.Cancer Discov. 2016; 6: 1276-1291Crossref PubMed Scopus (102) Google Scholar]. GABPA forms a complex with BRD4 for epigenetic activation of hTERT where the GABPA–BRD4 complex induced a permissive chromatin conformation by increasing H3K4me2/3 and H3K9ac activation marks at the hTERT promoter [27.Akıncılar S.C. et al.Long-range chromatin interactions drive mutant TERT promoter activation.Cancer Discov. 2016; 6: 1276-1291Crossref PubMed Scopus (102) Google Scholar, 28.Stern J.L. et al.Allele-specific DNA methylation and its interplay with repressive histone marks at promoter-mutant TERT genes.Cell Rep. 2017; 21: 3700-3707Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29.Shanmugam R. et al.Genome-wide screens identify specific drivers of mutant hTERT promoters.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2105171119Crossref PubMed Scopus (3) Google Scholar]. Furthermore, the β1L isoform of GABP is most critical for telomerase hyperactivation in glioblastomas. GABP functions as a dimer/tetramer of its alpha and beta subunits; while the alpha subunit remains constant, the beta subunit can vary between β1 and β2. Of these, β1 can be long (β1L) or short (β1S). Mutation or deletion of GABPβ1L caused telomerase resuppression in hTERT promoter mutant glioblastoma [30.Mancini A. et al.Disruption of the β1L isoform of GABP reverses glioblastoma replicative immortality in a TERT promoter mutation-dependent manner.Cancer Cell. 2018; 34: 513-528.e8Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. Like GABP, other ETS binding factors mediate telomerase reactivation in cancer. Noncanonical NF-κB (p52) induced by TWEAK (tumor necrosis factor-like weak inducer of apoptosis) hyperactivates telomerase in −146G>A but not in −124G>A mutant glioblastomas. NF-κB signaling induces tumor proliferation in −146G>A mutant glioblastoma cells in vitro and T98G-derived xenografts in NOD-SCID mice in vivo. Furthermore, p52 binds in a cooperative manner with the ETS1/2 at the hTERT promoter. While both −124 and −146 mutant sites create novel ETS binding sites, they differ by the presence of a T (−146 CCCGACCCTTCCGGGTCCCC and −124 site GGCCCAGCCCTTCCGGGCCCTC) at the beginning of the ETS binding motif. The presence of TCC in the −146G>A mutation site creates specificity for p52 binding in cooperation with ETS. The presence of ETS at the mutant hTERT promoter is, however, essential for p52-mediated telomerase reactivation [31.Li Y. et al.Non-canonical NF-κB signalling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation.Nat. Cell Biol. 2015; 17: 1327-1338Crossref PubMed Scopus (137) Google Scholar,32.Tergaonkar V. NFκB drives TERT promoter reactivation in cancer.Cell Cycle. 2016; 15: 156-157Crossref PubMed Scopus (7) Google Scholar]. Furthermore, the crystal structure of p52/ETS1 in complex with the promoter segment harboring the −146C>T mutation revealed p52 in the homodimeric form. Along with ETS1, this finding suggests formation of a heterotetramer at the mutant hTERT promoter where the p52/ETS1 heterodimer interacts through p52 homodimerization to form a heterotetramer. This complex was also shown to interact with the native ETS binding motifs flanking the −146 mutation site within the hTERT promoter, much like the GABP heterotetramer [26.Bell R.J.A. et al.The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer.Science. 2015; 348: 1036-1039Crossref PubMed Scopus (343) Google Scholar,33.Xu X. et al.Structural basis for reactivating the mutant TERT promoter by cooperative binding of p52 and ETS1.Nat. Commun. 2018; 9: 3183Crossref PubMed Scopus (30) Google Scholar]. ETS1-mediated telomerase reactivation is found in melanoma cell lines. As such, silencing of ETS1 resulted in significant telomerase suppression [34.Vallarelli A.F. et al.TERT promoter mutations in melanoma render TERT expression dependent on MAPK pathway activation.Oncotarget. 2016; 7: 53127-53136Crossref PubMed Scopus (43) Google Scholar]. ETS1 transcriptional function is dependent on Thr38 phosphorylation of ETS1 by extracellular signal-regulated kinase (ERK). This dependency is a consequence of mitogen-activated protein kinase (MAPK) pathway activation, which associates with simultaneous BRAF or NRAS mutations in melanomas. BRAF mutations are commonly reported to co-occur with hTERT promoter mutations in melanomas and thyroid cancers [35.Liu X. et al.TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer.J. Clin. Endocrinol. Metab. 2014; 99: E1130-E1136Crossref PubMed Scopus (214) Google Scholar, 36.Xing M. et al.BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence.J. Clin. Oncol. 2014; 32: 2718-2726Crossref PubMed Scopus (455) Google Scholar, 37.Jin L. et al.BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients.Oncotarget. 2016; 7: 18346-18355Crossref PubMed Scopus (85) Google Scholar, 38.Dubbink H.J. et al.TERT promoter mutations and BRAF mutations are rare in sporadic, and TERT promoter mutations are absent in NF1-related malignant peripheral nerve sheath tumors.J. Neuro-Oncol. 2014; 120: 267-272Crossref PubMed Scopus (13) Google Scholar, 39.Shi X. et al.Association of TERT promoter mutation 1,295,228 C>T with BRAF V600E mutation, older patient age, and distant metastasis in anaplastic thyroid cancer.J. Clin. Endocrinol. Metab. 2015; 100: E632-E637Crossref PubMed Scopus (63) Google Scholar]. In addition to NF-κB, RAS-ERK mediates telomerase reactivation in BRAFV600E mutant melanomas. ERK2 is recruited to the mutant hTERT promoter in association with GABP or ETS1. Following this recruitment, ERK2 phosphorylates the transcription factor SP1 and abrogates HDAC1 occupancy, thereby inducing a permissive promoter chromatin conformation through gain in H3K9 acetylation histone [40.Li Y. et al.Activation of mutant TERT promoter by RAS-ERK signaling is a key step in malignant progression of BRAF-mutant human melanomas.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 14402-14407Crossref PubMed Scopus (63) Google Scholar] (Figure 2). In thyroid cancers devoid of GABP activity, the ETS factor ETV5 binds −124G>A mutant hTERT promoter to reactivate telomerase in cooperation with FOXE1 [11.Bullock M. et al.ETS factor ETV5 activates the mutant telomerase reverse transcriptase promoter in thyroid cancer.Thyroid. 2019; 29: 1623-1633Crossref PubMed Scopus (17) Google Scholar]. In hepatocellular carcinoma, prospero homeobox protein 1 (PROX1) transcriptionally activates the mutant hTERT promoter. However, this does not appear to be the main mechanism of telomerase reactivation, because hepatocellular carcinoma expresses high levels of the hepatitis B virus X (HBx) protein, which suppresses PROX1 expression in vitro [41.Kim Y.-J. et al.Suppression of PROX1-mediated TERT expression in hepatitis B viral hepatocellular carcinoma.Int. J. Cancer. 2018; 143: 3155-3168Crossref PubMed Scopus (9) Google Scholar]. Recently, transcription factor tripartite motif containing 28 (TRIM28) was found to induce transcriptional activation of telomerase in bladder cancers with hTERT promoter mutation (−124G>A). GABPA mediates the recruitment of TRIM28 along with TRIM24 at the mutant hTERT promoter, where TRIM24 represses telomerase. Phosphorylation of TRIM28 by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) releases TRIM24, resulting in hyperactivation of telomerase [42.Agarwal N. et al.TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2102423118Crossref Scopus (9) Google Scholar]. Figure 2 summarizes the different molecular mechanisms known to reactivate telomerase because of hTERT promoter mutations in cancers. Considering the recurrence of hTERT promoter mutations, several groups have explored the significance of telomerase reactivation in neoplastic transformation. hTERT promoter mutations have been reported as early events in cancer initiation [43.Wang N. et al.TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.Cancer. 2014; 120: 2965-2979Crossref PubMed Scopus (78) Google Scholar, 44.Cheng L. et al.Telomerase reverse transcriptase (TERT) promoter mutation analysis of benign, malignant and reactive urothelial lesions reveals a subpopulation of inverted papilloma with immortalizing genetic change.Histopathology. 2016; 69: 107-113Crossref PubMed Scopus (37) Google Scholar, 45.Shain A.H. et al.The genetic evolution of melanoma from precursor lesions.N. Engl. J. Med. 2015; 373: 1926-1936Crossref PubMed Scopus (599) Google Scholar] and have been studied as driver mutations rather than sporadic events [21.Horn S. et al.TERT promoter mutations in familial and sporadic melanoma.Science. 2013; 339: 959-961Crossref PubMed Scopus (1245) Google Scholar,22.Huang F.W. et al.Highly recurrent TERT promoter mutations in human melanoma.Science. 2013; 339: 957-959Crossref PubMed Scopus (1291) Google Scholar]. However, while essential for tumor progression, hTERT promoter mutations are clonal and therefore nonessential in glioma initiation [46.Stead L.F. Verhaak R.G.W. Doomed from the TERT? A two-stage model of tumorigenesis in IDH-wild-type glioblastoma.Cancer Cell. 2019; 35: 542-544Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. In another study, normal bladder stem cells transformed to bladder cancer cells following telomerase hyperactivation due to −124G>A promoter mutation. Conversely, correcting the −124G>A mutation back to G resuppressed telomerase in bladder cancer stem cells, resulting in inhibition of tumor proliferation [47.Li C. et al.The C228T mutation of TERT promoter frequently occurs in bladder cancer stem cells and contributes to tumorigenesis of bladder cancer.Oncotarget. 2015; 6: 19542-19551Crossref PubMed Scopus (34) Google Scholar]. Consistent with these findings, when normal somatic cells, which generally lack telomerase, are introduced with hTERT promoter mutations, telomerase is reactivated and cell proliferation is induced [9.Chiba K. et al.Cancer-associated TERT promoter mutations abrogate telomerase silencing.eLife. 2015; 4e07918Crossref Scopus (175) Google Scholar], suggesting that telomerase reactivating mutations can induce immortalization of normal somatic cells [9.Chiba K. et al.Cancer-associated TERT promoter mutations abrogate telomerase silencing.eLife. 2015; 4e07918Crossref Scopus (175) Google Scholar]. This finding was later recognized as a two-step process in melanoma: (i) hTERT promoter mutations reactivate telomerase on critically short telomeres that are barely maintained to prevent inhibition of cell proliferation and thereby extend cellular lifespan, and (ii) in the second step, critically short telomeres induce genomic instability and hTERT promoter mutations further enhance telomerase to elongate telomeres and sustain cell proliferation [48.Chiba K. et al.Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism.Science. 2017; 357: 1416-1420Crossref PubMed Scopus (156) Google Scholar, 49.Pestana A. et al.TERT biology and function in cancer: beyond immortalisation.J. Mol. Endocrinol. 2017; 58: R129-R146Crossref PubMed Scopus (52) Google Scholar, 50.Heidenreich B. Kumar R. TERT promoter mutations in telomere biology.Mutat. Res. 2017; 771: 15-31Crossref PubMed Scopus (106) Google Scholar]. In overall agreement with these hTERT mechanisms, hTERT promoter mutations are reported as diagnostic biomarkers in several cancers, including bladder, thyroid, renal, and pelvic cancer; glioma; and spitzoid melanocytic neoplasms [43.Wang N. et al.TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.Cancer. 2014; 120: 2965-2979Crossref PubMed Scopus (78) Google Scholar,51.Allory Y. et al.Telomerase reverse transcriptase promoter mutations in bladder cancer: high frequency across stages, detection in urine, and lack of association with outcome.Eur. Urol. 2014; 65: 360-366Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 52.Hurst C.D. et al.Comprehensive mutation analysis of the TERT promoter in bladder cancer and detection of mutations in voided urine.Eur. Urol. 2014; 65: 367-369Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 53.Wang Z. et al.Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E6293-E6300Crossref PubMed Scopus (64) Google Scholar, 54.Wang K. et al.TERT promoter mutations are associated with distant metastases in upper tract urothelial carcinomas and serve as urinary biomarkers detected by a sensitive castPCR.Oncotarget. 2014; 5: 12428-12439Crossref PubMed Scopus (46) Google Scholar, 55.Wang X. et al.Association of telomerase reverse transcriptase promoter mutations with the prognosis of glioma patients: a meta-analysis.Mol. Neurobiol. 2016; 53: 2726-2732Crossref PubMed Scopus (9) Google Scholar, 56.Lee S. et al.TERT promoter mutations are predictive of aggressive clinical behavior in patients with spitzoid melanocytic neoplasms.Sci. Rep. 2015; 5: 11200Crossref PubMed Scopus (99) Google Scholar;reviewed in 57.Vinagre J. et al.Telomerase promoter mutations in cancer: an emerging molecular biomarker?.Virchows Arch. 2014; 465: 119-133Crossref PubMed Scopus (90) Google Scholar]. hTERT promoter mutations significantly correlate with old age across gliomas, central nervous system-associated cancers, thyroid cancers, and hepatocellular carcinoma [58.Eckel-Passow J.E. et al.Glioma groups based on 1p/19q, IDH, and TERT promoter mutations in tumors.N. Engl. J. Med. 2015; 372: 2499-2508Crossref PubMed Scopus (1268) Google Scholar, 59.Jang J.-W. et al.Significance of TERT genetic alterations and telomere length in hepatocellular carcinoma.Cancers (Basel). 2021; 13: 2160Crossref PubMed Scopus (7) Google Scholar, 60.Liu T. et al.The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas.Oncogene. 2014; 33: 4978-4984Crossref PubMed Scopus (172) Google Scholar, 61.Vinagre J. et al.Frequency of TERT promoter mutations in human cancers.Nat. Commun. 2013; 4: 2185Crossref PubMed Scopus (607) Google Scholar]. Particularly, co-occurrence of hTERT promoter mutations along with BRAFV600E mutations in thyroid cancer is associated with aging [39.Shi X. et al.Association of TERT promoter mutation 1,295,228 C>T with BRAF V600E mutation, older patient age, and distant metastasis in anaplastic thyroid cancer.J. Clin. Endocrinol. Metab. 2015; 100: E632-E637Crossref PubMed Scopus (63) Google Scholar], and the −146G>A mutation specifically correlates with telomere length in head and neck cancers [62.Barczak W. et al.hTERT C250T promoter mutation and telomere length as a molecular markers of cancer progression in patients with head and neck cancer.Mol. Med. Rep. 2017; 16: 441-446Crossref PubMed Scopus (14) Google Scholar]. In patients with long telomeres, point mutations in the hTERT promoter appear to correlate with resistance to radiotherapy and poor survival [63.Gao K. et al.TERT promoter mutations and long telomere length predict poor survival and radiotherapy resistance in gliomas.Oncotarget. 2016; 7: 8712-8725Crossref PubMed Scopus (52) Google Scholar]. In addition, a more direct interaction between telomere length and telomerase regulation has been reported. The hTERT promoter locus (1.2 Mb from telomeres on chromosome 5p) associates with relatively long telomeres through chromatin looping, which results in telomere-like heterochromatin formation at the promoter, leading to hTERT suppression [64.Kim W. et al.Regulation of the human telomerase gene TERT by telomere position effect-over long distances (TPE-OLD): implications for aging and cancer.PLoS Biol. 2016; 14e2000016Crossref Scopus (106) Google Scholar]. Consistent with this finding, normal aged fibroblasts express higher levels of hTERT because they lack looping-mediated chromatin interactions due to shorter telomeres. Association of the telomere repeat binding factor 2 (TRF2) at the hTERT promoter locus is a result of telomeric looping (Figure 3A ); however, whether other telomere-binding proteins, such as TRF1, POT1, or RAP1, also associate with the hTERT promoter due to looping is not clear. Independently, telomere-binding proteins TRF2 and NM23-H2 have been reported to interact with the promoter and affect hTERT transcription in cancers [65.Sharma S. et al.Human telomerase expression is under direct transcriptional control of the telomere-binding-factor TRF2.bioRxiv. 2020; (Published online January 15, 2020)https://doi.org/10.1101/2020.01.15.907626Google Scholar,66.Saha D. et al.Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.J. Biol. Chem. 2017; 292: 15205-15215Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. In cancers with hTERT promoter mutations, a GABPA binding site approximately 300 kb upstream (known as the T-INT1 locus) is associated with the hTERT promoter through chromatin looping [27.Akıncılar S.C. et al.Long-range chromatin interactions drive mutant TERT promoter activation.Cancer Discov. 2016; 6: 1276-1291Crossref PubMed Scopus (102) Google Scholar] (Figure 3B). This finding further substantiates the finding that GABPA mediates BRD4 recruitment on the mutant hTERT promoter, which results in permissive chromatin conformation [27.Akıncılar S.C. et al.Long-range chromatin interactions drive mutant TERT promoter activation.Cancer Discov. 2016; 6: 1276-1291Crossref PubMed Scopus (102) Google Scholar]. While GABPA occupancy at the T-INT1 region was shown in cells without prom